SummaryWe previously identified HpuB, an 85 kDa Fe-repressible protein required for utilization of Fe from, and binding to, haemoglobin and the haemoglobin-haptoglobin complex. The gene for hpuB was cloned from Neisseria meningitidis strain DNM2 and the predicted amino acid sequence indicates that HpuB is an outer membrane receptor belonging to the TonB family of high-affinity transport proteins. A second open reading frame, predicted to encode a 34.8 kDa lipoprotein, was discovered 5Ј to hpuB, and was designated hpuA. HpuA was identified in a total-membrane-protein preparation by construction of a mutant lacking HpuA. Acylation of HpuA was confirmed by [ 3 H]-palmitic acid labelling of meningococci. Consensus promoter sequences were not apparent 5Ј to hpuB. The hpuA insertion mutation exerted a polar effect, abolishing expression of hpuB, suggesting that hpuA and hpuB are co-transcribed. The 3.5 kb polycistronic hpuAB mRNA was identified and shown to be transcriptionally repressed by iron. The transcriptional start site was identified 33 nucleotides 5Ј to the hpuA translational start site, appropriately positioned around consensus promoter and ferric uptake regulator (Fur)-box sequences. The structure of this operon suggests that HpuA-HpuB is a two-component receptor analogous to the bipartite transferrin receptor TbpB-TbpA.

An important event in the development of cervical squamous cell carcinoma (SCC) is deregulated expression of high-risk human papillomavirus (HR-HPV) oncogenes, most commonly related to viral integration into host DNA. Mechanisms of development of the ∼15% of SCCs that contain extrachromosomal (episomal) HR-HPV are poorly understood due to limited longitudinal data. We therefore used the W12 model to study mechanisms of cervical carcinogenesis associated with episomal HPV16. In vitro progression of W12 normally occurs through selection of cells containing integrated HPV16. However, in one long-term culture, keratinocytes developed a selective growth advantage and invasive phenotype while retaining HPV16 episomes at increased copy number in the absence of transcriptionally active integrants. Longitudinal investigations revealed similarities between the episome-and integrant-associated routes of neoplastic progression. Most notable were dynamic changes in viral early gene expression in episome-retaining cells, consistent with continually changing selective pressures. An early increase in viral transcription preceded elevated episome copy number and was followed by a reduction to near baseline after the development of invasiveness. Episomal transcriptional deregulation did not require selection of a specific sequence variant of the HPV16 upstream regulatory region, although increased levels of acetylated histone H4 around the late promoter implicated a role for altered chromatin structure. Interestingly, invasive episome-retaining cells showed high levels of HPV16 E2/ E6 proteins (despite decreased transcript levels) and reduced expression of IFN-stimulated genes, adaptations that support viral persistence and cell survival. Our findings suggest a unified working model for events important in cervical neoplastic progression regardless of HR-HPV physical state. Cancer Res; 70(10); 4081-91.

+ Tregs constitute a heterogeneous lymphocyte subpopulation essential for curtailing effector T cells and establishing peripheral tolerance. Calcineurin inhibitors (CNIs) are among the most effective agents in controlling effector T-cell responses in humans. However, CNIs also reduce the size of the Treg pool. The functional consequences of this negative effect and the mechanisms responsible remain to be elucidated. We report here that CNIs compromise the overall Treg immunoregulatory capacity to a greater extent than would be predicted by the reduction in the size of the Treg compartment, given that they selectively promote the apoptosis of the resting and activated Treg subsets that are known to display the most powerful suppressive function. These effects are caused by reduced access to IL-2, because Tregs remain capable of translocating NFAT even in the presence of high CNI levels. Exogenous IL-2 restores the phenotypic changes and overall gene-expression effects exerted by CNIs and can even promote Treg expansion by enhancing antiapoptotic Bcl-2 expression. In a skin transplant model, the addition of IL-2 synergizes with CNIs treatment, promoting intragraft accumulation of Tregs and prolonged allograft survival. Hence, the combination of IL-2 and CNIs constitutes an optimal immunomodulatory regimen that enhances the pool of suppressive Treg subsets while effectively controlling cytopathic T cells.regulatory T cells | transplantation | IL-2 therapy | calcineurin inhibitors |

The extracellular calcium-sensing receptor (CaR) is usually associated with systemic Ca 2C homeostasis, but the CaR is also expressed in many other tissues, including pancreatic islets of Langerhans. In the present study, we have used human islets and an insulin-secreting cell line (MIN6) to investigate the effects of CaR activation using the calcimimetic R-568, a CaR agonist that activates the CaR at physiological concentrations of extracellular Ca 2C . CaR activation initiated a marked but transient insulin secretory response from both human islets and MIN6 cells at a sub-stimulatory concentration of glucose, and further enhanced glucose-induced insulin secretion. CaR-induced insulin secretion was reduced by inhibitors of phospholipase C or calcium-calmodulindependent kinases, but not by a protein kinase C inhibitor. CaR activation was also associated with an activation of p42/44 mitogen-activated protein kinases (MAPK), and CaR-induced insulin secretion was reduced by an inhibitor of p42/44 MAPK activation. We suggest that the b-cell CaR is activated by divalent cations co-released with insulin, and that this may be an important mechanism of intra-islet communication between b-cells.

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Details concerning the endogenous regulation of hepatic cytochrome P450 monooxygenases in teleosts, and the features of this regulation common among fish species, are poorly known. Gonadally mature female winter flounder (Pseudopleuronectes americanus) have been reported to have severalfold lower levels of microsomal cytochromes P450 and b5 and NADPH-cytochrome c reductase than do males (Stegeman and Woodin ('84) Mar. Environ. Res., 14:422-425). These strong sex differences prompted more detailed study of P450 regulation in winter flounder liver, and a comparison with sex differences in another marine teleost, scup (Stenotomus chrysops). Ethoxyresorufin O-deethylase (EROD) activity/nmol P450 was less in gonadally mature females than in males of both species. Immunoblot analysis with MAb 1-12-3 to P450E (the EROD catalyst) showed that the content of P450E counterpart was also much less in females of both species. Aminopyrine N-demethylase (APND) and testosterone 6 beta-hydroxylase (6 beta-OHase) activities per nmol P450 were higher in gonadally mature female than in mature male flounder, differences not seen in scup. Polyclonal antibodies to scup P450A were shown to detect proteins in a number of teleosts. The levels of anti-P450A cross-reacting protein were greater in mature female than in male flounder, but as with 6 beta-OHase activity, the content of this protein was not sexually differentiated in scup. Estradiol treatment of winter flounder depressed the rates of EROD, APND, 6 beta-OHase, and estradiol 2-OHase activities per mg protein, but APND and 6 beta-OHase activities per nmol P450 were unchanged. Thus, E2 promotes general decreases in some hepatic P450-catalyzed activities, but in achieving sex differences there is also specific regulation of the P450E counterpart, and possibly of the 6 beta-OHase (P450A?). Other factors, temporal or hormonal, can modify the effect of E2 treatment, and may contribute to the specific regulation of P450 forms in naturally maturing fish, and to species differences in this regulation.

Binding sites for native chum salmon growth hormone (sGH) in coho salmon (Oncorhynchus kisutch) hepatic membranes were analyzed by radioreceptor assay. Displaceable (specific) binding represented up to 25% of total radiolabeled sGH added. Binding was dependent on buffer pH and membrane protein concentration, and was complete by 24 hours at 15 degrees C. Specific binding was greatest in liver membranes, and was also detected in muscle, ovary, gill, kidney, and brain. Scatchard analyses indicated a single class of hepatic binding sites that were specific for sGH. In stunts, abnormal seawater salmon with elevated plasma GH levels and inhibited growth, specific binding of sGH to liver membranes was three times lower than in normal seawater smolts. The concentration of salmon GH binding sites was decreased in stunt livers by 60%, while their affinity for sGH was unchanged. Down-regulation of hepatic GH receptors by high plasma GH levels may explain in part the low sGH binding in stunts.